Skeletal and craniofacial diseases affect millions of patients each year in the U.S. Identifying the function and mechanism of the new genes in craniofacial and skeletal development and homeostasis is critical for treatment of these diseases. Intraflagellar transport (IFT) protein is essential for cilia formation and crucial for processing of many signaling components such as Hedgehog and Gli signaling. It is known that Cilia-related proteins regulate craniofacial and bone development and mechanically regulate bone homeostasis in adults. However, how IFT protein regulates OB differentiation and function during those processes remains largely unknown. IFT80 is a newly-identified IFT protein. Reduced expression of IFT80 in humans causes Jeune asphyxiating thoracic dystrophy and short rib polydactyly type III with very severe bone abnormalities. However, the mechanism by which IFT80 functions in skeletal development and homeostasis is unknown. Our in vitro studies suggest that IFT80 plays an important role in osteogenesis. By deleting IFT80 in osteoblast (OB) specific lineage using Osterix-cre, we found that IFT80flox/flox/Osx-cre mice showed apparent growth retardation with severe tooth and craniofacial bone abnormalities and significant decrease in bone mass. Deletion of IFT80 impaired cilia formation and OB differentiation accompanied by down-regulated Hh-Gli signaling activities. Most importantly, deletion of IFT80 significantly inhibited Runx2 and BMP2 gene expression during OB differentiation. Overexpression of Gli2 or these two genes could rescue defective OB differentiation in IFT80 deleted cells. Thus, we hypothesize that IFT80 is required for cilia formation and OB differentiation and that loss of IFT80 results in the inability of pre-OBs and mature OBs to maintain their stage of differentiation, resulting in cells responding abnormally to mechanotransduction and altering skeletal development and homeostasis. We will test the hypothesis through three Specific Aims. In Aim 1. We will reveal the in vivo function of IFT80 at various stages of OB differentiation by analyzing bone phenotypes of IFT80flox/flox/Osx-cre and inducible IFT80flox/flox/col1a1-cre and determining whether deletion of IFT80 affects mechanical loading caused bone formation. We will further define whether mutation of different IFT gene causes different bone phenotype by comparing IFT80flox/flox/Osx-cre and IFT20flox/flox/Osx-cre mice. In Aim 2. We will elucidate in vitro the role and mechanism by which IFT80 regulates of OB differentiation at different stages by performing the studies with IFT80del cells and lentivirus mediated RNAi and retrovirus mediated overexpression with or without fluid flow shear stress stimulation. We will further investigate the mechanism by which IFT80 regulates transcription factors and critical signaling proteins in OB differentiation and mechanosensary function, with particular emphasis on its interplay with Hh, Gli, BMP2 and Runx2 signaling and IFT80 downstream genes. In Aim 3. We will dissect the molecular mechanism of IFT80 interactions that confers cilia formation and OB differentiation and function by characterizing IFT80 structural domains, interacting proteins and their functions.